The long-term goals of this proposal are to define the molecular mechanisms that regulate the spatial distribution of organelles in the early secretory pathway and to determine the importance of this architecture to normal membrane trafficking during cell growth and development. Most biosynthetic cargo molecules destined for secretion initiate their journey within specific subdomains of the endoplasmic reticulum (ER), known as ER exit sites. At these locations, COPII-coated vesicles are first generated, packaging cargoes for transport to ER- Golgi intermediate compartments (ERGIC), stable organelles that are juxtaposed to ER exit sites. The COPII coat is composed of two multimeric protein complexes, Sec23/24 and Sec13/31, and the small GTPase Sar1. Although these factors are sufficient to reconstitute vesicle budding from chemically defined membranes in vitro, additional proteins are required to promote COPII vesicle biogenesis and anterograde transport in cells. This proposal focuses on the role of TFG, a metazoan-specific protein required for the robust recruitment of COPII coat subunits to ER exit sites. Based on our preliminary results, we hypothesize that TFG forms a highly regulated meshwork at the ER/ERGIC interface that facilitates COPII coat stability and vesicle egress. Importantly, mutations in TFG have been implicated in progressive neurodegenerative disease, suggesting a role for COPII-mediated transport in maintaining neuron function. We propose a combination of in vivo and in vitro approaches to achieve our aims, taking advantage of assays developed in human cells and biochemical methodologies established to study the structure and function of early secretory pathway components. Using electron tomography, we recently defined the architecture of the early secretory pathway in germ cells derived from the model organism C. elegans. Our findings revealed the presence of an electron-dense meshwork, filled with molecules of TFG, which encompasses the region between ER exit sites and ERGIC membranes. We hypothesize that this meshwork functions in the regulation of COPII dynamics and helps to maintain the organization of the early secretory pathway. In a similar fashion, our preliminary studies in human cells have demonstrated a conserved role for TFG in controlling early secretory pathway architecture and function. Additionally, we have recently defined a structural model for TFG using cryo-electron microscopy and small angle X-ray scattering, which has led us to propose a testable model for the role of TFG at the interface between ER and ERGIC membranes. The specific aims of this proposal are to: 1) determine the structural basis for TFG assembly and disassembly at the ER/ERGIC interface, 2) define the contributions of TFG to COPII-mediated vesicle transport, and 3) define mechanisms by which TFG contributes to neuronal maintenance. Together, the experiments outlined in this proposal will provide fundamental new insights into how the organization of the early secretory pathway promotes the rapid anterograde transport of newly synthesized cargoes in COPII-coated vesicles, which is necessary for normal human development and neuronal homeostasis.